CN112724212B - Application of quinoa protein in resisting plant germs - Google Patents

Abstract

The invention provides an application of quinoa protein in resisting plant germs, wherein the quinoa protein is prepared by the following steps: crushing quinoa seeds, degreasing, leaching, carrying out ammonium sulfate fractional precipitation, carrying out filtration chromatography and two-step chromatographic separation and purification by using a DEAE-FF anion exchange column and a Sephacry S-200 gel column to obtain high-purity quinoa protein, and identifying that the amino acid sequence of the quinoa protein has certain similarity with a part of plant 1, 5-diphosphoribulose carboxylase/oxygenase large subunits with different sources by mass spectrometry. The protein has obvious effect of inhibiting Alternaria tenuissima (Alternaria tenuissima) and also has certain inhibiting effect on Fusarium solani (Fusarium solani). The antibacterial protein has simple preparation process, can be used as a plant anti-bacterial preparation and the like, and is beneficial to the development of quinoa protein resources and the application in the fields of agriculture and the like.

Description

Application of quinoa protein in resisting plant germs

Technical Field

The invention relates to preparation and application of vegetable protein, in particular to preparation of quinoa protein and application of quinoa protein in resisting plant germs.

Background

Chenopodium quinoa willd, also known as Chenopodium quinoa willd, Indian wheat, and fructus Rosae Laevigatae. Belonging to the genus Chenopodium of the family Chenopodiaceae. Native to Andes mountain of south America, and has a planting history of 5000-7000 years. Because of its rich nutritive value, the ancient signer is called the "mother of grain" (mothers grain) to breed the signer. Quinoa is rich in complete protein and various amino acids, wherein 9 amino acids necessary for human bodies are contained, particularly lysine which is deficient in plants is contained, and the chemical components are closely related to the functions of supplementing nutrition, enhancing body functions, repairing physique, improving body immunity, preventing diseases, resisting cancers and the like. At present, most of researches focus on active ingredients such as quinoa polyphenol, flavone and saponin, but the researches on the protein aspect are few, and especially the researches on the anti-bacterial activity aspect are not reported yet.

Alternaria Nees is distributed worldwide, has saprophytic, endogenous and pathogenic properties, can infect and harm various crops, can cause diseases alone, and can also infect a host together with other pathogenic bacteria. The alternaria fungus has strong environment adaptability, can cause mildew in the fields, transportation and storage processes, has large variety grade characteristic variation, and has very difficult disease control due to different infection modes and different ecological environments of pathogenic mechanisms. Alternaria tenuissima (Alternaria tenuissima) is one of the main pathogenic bacteria causing black rot of crops, especially jujube. Fusarium solani is a fungus that produces toxins, causing toxicity, canceration and parasitic thallus in animals and humans, resulting in abortion, corneal and skin ulcers.

Disclosure of Invention

The invention aims to provide a preparation method of quinoa protein and application of quinoa protein in resisting plant germs.

In order to realize the purpose, the technical scheme is as follows:

the invention provides an application of quinoa protein in resisting plant germs, wherein the quinoa protein is prepared by the following steps:

(1) crushing quinoa seeds, degreasing, weighing a certain amount of degreased quinoa powder, and mixing the powder according to a material-liquid ratio (W/V) 1: dissolving 5-20 in 20mmol/L acetic acid-ammonium acetate buffer solution with pH of 4.5, stirring and leaching at 4 deg.C for 10-20 hr, centrifuging the above sample at 12000r/min for 30min, collecting supernatant, removing precipitate to obtain crude extract of quinoa seed protein;

(2) slowly adding a certain amount of ammonium sulfate with the saturation degree of 80% into the crude extract at 4 ℃, stirring while adding, placing into a refrigerator at 4 ℃ after the ammonium sulfate is completely dissolved, standing for precipitation for more than 4h, centrifuging at 12000r/min for 30min, discarding the supernatant, dissolving the precipitate with 20mmol/L, pH 7.0.0 PBS buffer solution, dialyzing with the same buffer solution at 4 ℃ for 20h, replacing the buffer solution for 3-4 times, centrifuging at 12000r/min at 4 ℃ for 30min, and collecting the supernatant;

(3) loading the supernatant into an AKTA PURIFIER protein purification system, balancing a DEAE-FF anion column by using 20mmol/L, pH-7.0 PBS buffer solution in advance, loading 20mL of buffer solution each time, eluting with the same buffer solution at the flow rate of 2mL/min, washing off unadsorbed protein, eluting with the buffer solution containing 0.5mol/L NaCl at the flow rate of 2mL/min, and collecting 30% of eluted components;

(4) dialyzing the elution component obtained after DEAE-FF anion exchange with 20mmol/L, pH 7.0.0 PBS buffer solution, concentrating by using a concentration tube, loading on a Sephacryy S-200 gel filtration chromatographic column which is balanced by 20mmol/L, pH 7.0.0 PBS buffer solution in advance, loading 5mL of elution component each time at the flow rate of 1mL/min, collecting the elution peak, and freeze-drying to obtain the quinoa protein.

The feed-liquid ratio in the step (1) is preferably 1: 15; the agitation leaching time is preferably 14.6 hours.

The plant pathogen is preferably Alternaria tenuissima or Fusarium solani.

Compared with the prior art, the invention has the beneficial effects that: the invention aims to provide a novel quinoa protein with anti-bacterial activity, which is obtained by separating and purifying quinoa seeds through column chromatography based on that quinoa seeds contain abundant complete protein and various amino acids. The quinoa protein prepared by the method is convenient to amplify in production by using a simple separation and purification method, and has a remarkable antibacterial effect on plant pathogenic bacteria, particularly Alternaria tenuissima and Fusarium solani. The invention is beneficial to the development and application of quinoa resources in the fields of pesticides, medicines and the like.

Drawings

FIG. 1 is a DEAE-FF anion exchange chromatography elution profile;

FIG. 2 is an SDS-PAGE image of the purification and separation process; wherein: 1. crude protein, 2, total protein after desalting, 3, protein samples obtained after DEAE anion column gradient elution, 4, 20% protein samples obtained after gradient elution, 5, 30% protein samples obtained after gradient elution, 6, protein samples obtained after Sephacryl S-200 column gel chromatography;

FIG. 3 is a graph showing the results of mass spectrometric identification;

FIG. 4 alignment of amino acid sequences of quinoa proteins with portions of plant 1, 5-diphosphoribulose carboxylase/oxygenase large subunit amino acid sequences of different origins;

FIG. 5 is a graph showing the results of inhibition of Alternaria tenuissima (Alternaria tenuissima) by quinoa protein;

FIG. 6 is a graph showing the results of inhibition of Fusarium solani (Fusarium solani) by quinoa protein.

Detailed Description

Example 1

The first step is as follows: crushing quinoa seeds into powder, degreasing with petroleum ether, accurately weighing 30g of degreased quinoa powder, and mixing the powder according to a material-liquid ratio of 1: 15 is dissolved in 20mmol/L ammonium acetate-acetic acid buffer solution with pH4.5, stirred and leached for 14.6h at 4 ℃, then the sample is centrifuged for 30min at 12000r/min, and supernatant is collected and sediment is discarded, thus obtaining the crude extract of the quinoa seed protein;

the second step is that: slowly adding saturated ammonium sulfate 80% into the crude extractive solution, settling, standing overnight, centrifuging at 12000r/min for 30min, discarding supernatant, dissolving the precipitate with 20mmol/L PBS buffer solution with pH7.0, dialyzing with the same buffer solution at 4 deg.C for 20 hr, changing buffer solution for 3 times, centrifuging at 12000r/min at 4 deg.C for 30min, and collecting supernatant;

the third step: loading the supernatant into AKTA PURIFIER protein purification system, pre-balancing DEAE-FF anion column with 20mmol/L, pH of 7.0PBS buffer solution, loading 20mL each time, eluting with the same buffer solution at flow rate of 2mL/min, washing off unadsorbed protein (penetrating component A1), gradient eluting with the above buffer solution containing 0.5mol/L NaCl at flow rate of 2mL/min to obtain component A2 at 20% gradient elution, obtaining component A3 at 30% gradient elution, and collecting penetrating component A1, eluting components A2 and A3, respectively, as shown in FIG. 1.

The fourth step: respectively dialyzing the elution components A2 and A3 obtained after DEAE-FF anion exchange with the same buffer solution as the dialysis step, concentrating different components by using a concentration tube, loading the components A2 and A3 on a Sephacryy S-200 gel filtration chromatographic column which is balanced by 20mmol/L, pH 7.0.0 PBS buffer solution in advance, loading 5mL of the component at each time at the flow rate of 1mL/min, collecting corresponding components, and identifying that the component A3 only contains one protein band through SDS-PAGE, wherein the quinoa anti-viral protein is shown in FIG. 2.

The fifth step: performing mass spectrum identification on the protein corresponding to the single band, wherein the sequence of the N-terminal peptide segment is determined as follows: (R. PNGLLLHIHR.A). The amino acid sequence of the protein has certain similarity with the large subunit of the 1, 5-diphosphoribulose carboxylase/oxygenase from plants with different parts of sources, and the molecular weight of the protein is 50kDa, as shown in figures 3 and 4.

And a sixth step: the inhibition effect of quinoa anti-pathogen proteins with different concentrations on plant pathogens such as Fusarium solani (Fusarium solani), Fusarium acuminatum (Fusarium acuminatum), Alternaria tenuis (Alternaria tenuissima), Phoma capitatum (Phoma glomerata), Fusarium oxysporum (Fusarium oxysporum f.sp.culimerinum) and the like is detected by a filter paper method.

Inoculating the tested fungus on a PDA flat plate, carrying out inverted culture at 27 ℃ until the diameter of the fungus is about 2 cm, carefully placing sterilized filter paper sheets on a culture medium by using tweezers, dropwise adding sample liquid with different dilution concentrations of 20 mu l onto the filter paper sheets, taking the sample liquid as a contrast, and taking out the sample for photographing when the fungus growth can be observed to inhibit the growth phenomenon.

FIG. 5 shows that quinoa protein has a significant inhibitory effect on Alternaria tenuissima, wherein 1,2, 3 and 4 respectively show that when quinoa protein with different concentrations (0,5,10 and 15mg/mL) is dripped onto a filter paper sheet, hyphae can continue to grow around the filter paper sheet after two or three days of culture, and the whole culture medium is circular and has regular edges. Quinoa protein with different concentrations is dripped into the filter paper sheet, and the solution permeates into the culture medium, so that the growth of nearby hyphae is inhibited, and obvious inhibition zones appear around the hyphae, as shown in 2, 3 and 4 in figure 5.

FIG. 6 shows the inhibition effect of quinoa protein on Fusarium solani (Fusarium solani), wherein 1,2, 3 and 4 respectively show that quinoa protein with different concentrations (0,1,2 and 4mg/mL) is dripped on a filter paper sheet, and the cultured bacteriostasis result graph shows that quinoa protein with smaller concentration also has the effect of inhibiting plant pathogenic bacteria.

Claims (1)

1. The application of the quinoa protein in resisting plant germs is characterized in that the quinoa protein is prepared by the following steps and methods:

(1) crushing quinoa seeds, degreasing, weighing a certain amount of degreased quinoa powder, and mixing the powder according to a material-liquid ratio (W/V) 1: 15 is dissolved in 20mmol/L acetic acid-ammonium acetate buffer solution with pH value of 4.5, stirred and leached for 14.6 hours at 4 ℃, then the sample is centrifuged for 30 minutes at 12000r/min, and supernatant is collected and sediment is discarded, thus obtaining the crude extract of the quinoa seed protein;

(2) slowly adding a certain amount of ammonium sulfate with the saturation degree of 80% into the crude extract at 4 ℃, stirring while adding, placing into a refrigerator at 4 ℃ after the ammonium sulfate is completely dissolved, standing for precipitation for more than 4h, centrifuging at 12000r/min for 30min, discarding the supernatant, dissolving the precipitate with 20mmol/L, pH 7.0.0 PBS buffer solution, dialyzing with the same buffer solution at 4 ℃ for 20h, replacing the buffer solution for 3-4 times, centrifuging at 12000r/min at 4 ℃ for 30min, and collecting the supernatant;

(3) loading the supernatant into an AKTA PURIFIER protein purification system, balancing a DEAE-FF anion column by using 20mmol/L, pH-7.0 PBS buffer solution in advance, loading 20mL of buffer solution each time, eluting with the same buffer solution at the flow rate of 2mL/min, washing off unadsorbed protein, eluting with the buffer solution containing 0.5mol/L NaCl at the flow rate of 2mL/min, and collecting 30% of eluted components;

(4) dialyzing the elution component obtained after the DEAE-FF anion exchange with 20mmol/L, pH 7.0.0 PBS buffer solution, concentrating by using a concentration tube, loading on a Sephacryl S-200 gel filtration chromatographic column which is well balanced by 20mmol/L, pH 7.0.0 PBS buffer solution in advance, loading 5mL of sample each time at the flow rate of 1mL/min, collecting the elution peak, and freeze-drying to obtain the quinoa protein;

the plant pathogenic bacteria are Alternaria tenuissima or Fusarium solani.

Images